Traditional protein methods for the typing of blood and semen are based on allelic differences in proteins (Divall, Electrophoresis 6:249-258, 1985; Sensabaugh, Current Topics in Biological and Medical Research 6:247-282, Alan R. Liss, New York, 1982). Enzyme and antigenic variants which have been examined in blood include adenylate deaminase, adenylate kinase, carbonic anhydrase I, erythrocyte acid phosphatase, esterase D, glyoxylase I, hemoglobin, peptidase A, phosphoglucomutase, and Gm, Lewis, and Rh antigens. In paternity testing, typing of the HLA antigens is commonly used (Terasaki, J. Family Law 16:543, 1977-1978). The analysis of proteins in semen involves the characterization of the following systems: ABO blood group, phosphoglucomutase, glyoxylate, peptidase A, and the Lewis antigens. While these traditional methods, when used in combination, can exclude up to 99% of the general population as the source of a biological sample, these methods do not allow the unequivocal identification of a particular individual as the source of the biological sample or as the first degree relation (Gaensslen, Sourcebook in Forensic Serology, Immunology and Biochemistry. U.S. Government Printing Office, Washington, D.C. 1983). Potential problems with protein typing include changes in electrophoretic mobility with aging of the sample, and possible cross-reactivities and artifacts due to other materials in the evidential sample (wool, for instance).
Polymorphic regions of human DNA are also being used as a basis for the identification of human bio-forensic evidence specimens and in the establishment of both first degree and higher order biological family relationships. The technique for the examination of polymorphic DNA regions is called "DNA fingerprinting," "DNA profiling," or "DNA typing," and has clearly become the method of choice in these areas. DNA typing with appropriate probes allows the identification of genotypes that would not be expected to be duplicated within the entire human population.
DNA typing is based on the detection of intergenic regions of nuclear DNA that are composed of variable numbers of tandem repeats (VNTRs). VNTRs are also known as "hypervariable minisatellite restriction fragment length polymorphisms." These loci are found on a number of different chromosomes, which makes the appearance of a given VNTR allele statistically independent of most others. The large number of alleles (up to 70-80 or more) that can be found at many of the VNTR loci, combined with the high level of heterozygosity (greater than 88%), are features that make VNTRs useful for identification purposes (Nakamura et al., Science 235:1616-1622, 1987).
For example, Jeffreys et al. have described the use of three VNTR-specific probes for DNA fingerprinting (Nature 316:76-80, 1985). The probes in the Jeffreys et al. study detected upwards of 15 hybridizing bands per person. Although the hybridization patterns were complex, this work led to results which convinced the British immigration authorities that a man desiring entry into the country was, in fact, the son of a (female) citizen (Jeffreys et al. Nature 317:818-819, 1985).
DNA fingerprinting using the DNA-print.TM. system (Lifecodes Corporation, Elmsford, N.Y.) with probes recognizing four different VNTR loci has also been used to establish paternity with a probability of greater than 99% (Baird et al., in Advances in Forensic Haemogenetics 2, Springer Verlag, New York, 1987).
Forensic applications of DNA fingerprinting have also been described. Gill et al. (Nature 318:577-579, 1985), successfully isolated DNA suitable for fingerprinting from such specimens as vaginal swabs, 4-yr-old dried bloodstains and semen stains on cloth. In another report on the DNA-print.TM. system, which allows discrimination at levels greater than in 10.sup.8 individuals, the probes were used to establish that human tissue found on a car apparently involved in a homicide was likely to be from the offspring of the parents of the car's registered owner, with a 1 in 160,000 chance that the tissue was derived from an unrelated individual (Baird et al., in Adv. Forensic Haemooenetics 2, Springer-Verlag, New York, 1987).
DNA typing presents obvious advantages over protein typing methodologies, including less interference from other materials in the evidential samples, and greater stability of DNA as compared to protein in dried samples. DNA typing also presents the possibility for re-use of the same DNA gel or blot for multiple sequential hybridizations. However, due to a lack of sensitivity of the probes, and the relatively small size of the useful restriction fragments identifying other VNTR loci, the true potential for the commercial application of such probes has not yet been realized.
The present invention provides a novel group of DNA probes that recognize the human VNTR locus, D4S139, a highly polymorphic region. These probes overcome the disadvantages briefly discussed above, while providing other related advantages.